The protective function of the epidermis depends critically on the normal differentiation of the keratinocytes. Cell to cell adhesion is essential in this process. Although desmosomes apparently provide the mechanical strength in adhesion, adherens junctions provide the specificity, and are essential for the proper formation of desmosomes as well. Cadherins are constituents of both desmosomes and adherens junctions. In this proposal, the principal investigator will examine the role of cadherins in epidermal differentiation. The principal investigator has three specific aims. The first is to examine the mechanism by which the classical, nondesmosomal cadherins regulate desmosome formation. The intracellular cytoskeleton binding domains of classical cadherins are essential for their function as adhesion molecules. Using a series of deletions in the intracellular domain, the principal investigator will determine their role in desmosome assembly. Mutants in the extracellular Ca-binding site will be used to examine whether Ca binding, and its consequence the cadherin- cadherin interaction, have a role. Using chimeric constructs, i.e., substituting the NCAM extracellular domain, the investigators will establish the relevance of the specificity of the interaction. The second specific aim is to define the regulation of stratification by desmosomal cadherins. For this, a dominant-negative desmoglein construct will be used to engineer keratinocytes without, or with greatly reduced number of desmosomes. The capacity for differentiation in vitro in such keratinocytes will be determined by following a set of differentiation markers. As an alternative approach, a more subtle way to block individual desmogleins, of which there are three, using antisense oligonucleotides will be attempted. The third specific aim is to investigate the function of cadherins in vivo creating three different lines of transgenic mice with cadherins targeted to the epidermis. First, a dominant-negative cadherin targeted to the epidermis in transgenic mouse model will be constructed. The dominant-negative construct is a previously characterized N-cadherin mutant containing a deletion of a large segment of the extracellular domain, but retaining the signal peptide, transmembrane, and cytoplasmic sequences. Targeting signals derive from the human K1 keratin gene, which confers suprabasal expression in transgenic animals. The construct was tested in transient transfections, and it was established that it is expressed and localised at the cell-call boundaries. In a separate set of experiments, use will be made of a knock-out missing the P-cadherin gene. This mouse has no epidermal phenotype, is healthy and fertile. Into this, using the same epidermal targeting, a functional P-cadherin will be added. The resulting mouse should have P-cadherin in the suprabasal layer, in contrast to normal skin where P-cadherin is basal. Finally, a different targeting vector, the one using the loricrin gene promoter shown to target expression in all layers of the epidermis, will be used to express N-cadherin. In transfection experiments N-cadherin expression had a profound effect on keratinocyte morphology, suggesting that cadherins not only influence the selectivity of cell-to-cell adhesion, but have a more instructive function in determining cell phenotypes.